Depletion of the C. elegans NAC Engages the Unfolded Protein Response, Resulting in Increased Chaperone Expression and Apoptosis Paul T. Arsenovic, Anthony T. Maldonado, Vaughn D. Colleluori, Tim A. Bloss* Department of Biology, James Madison University, Harrisonburg, Virginia, United States of America Abstract The nascent polypeptide-associated complex (NAC) is a highly conserved heterodimer important for metazoan development, but its molecular function is not well understood. Recent evidence suggests the NAC is a component of the cytosolic chaperone network that interacts with ribosomal complexes and their emerging nascent peptides, such that the loss of the NAC in chaperone-depleted cells results in an increase in misfolded protein stress. We tested whether the NAC functions similarly in Caeonorhabditis (C.) elegans and found that its homologous NAC subunits, i.e. ICD-1 and -2, have chaperone-like characteristics. Loss of the NAC appears to induce misfolded protein stress in the ER triggering the unfolded protein response (UPR). Depletion of the NAC altered the response to heat stress, and led to an up-regulation of hsp-4,a homologue of the human chaperone and ER stress sensor GRP78/BiP. Worms lacking both ICD-1 and the UPR transcription factor XBP-1 generated a higher proportion of defective embryos, showed increased embryonic apoptosis and had a diminished survival rate relative to ICD-1-depleted animals with an intact UPR. Up-regulation of hsp-4 in NAC-depleted animals was specific to certain regions of the embryo; in embryos lacking ICD-1, the posterior region of the embryo showed strong up-regulation of hsp-4, while the anterior region did not. Furthermore, loss of ICD-1 produced prominent lysosomes in the gut region of adults and embryos putatively containing lipofuscins, lipid/protein aggregates associated with cellular aging. These results are the first set of evidence consistent with a role for C. elegans NAC in protein folding and localization during translation. Further, these findings confirm C. elegans as a valuable model for studying organismal and cell-type specific responses to misfolded protein stress. Citation: Arsenovic PT, Maldonado AT, Colleluori VD, Bloss TA (2012) Depletion of the C. elegans NAC Engages the Unfolded Protein Response, Resulting in Increased Chaperone Expression and Apoptosis. PLoS ONE 7(9): e44038. doi:10.1371/journal.pone.0044038 Editor: Alejandro Aballay, Duke University Medical Center, United States of America Received June 15, 2012; Accepted August 1, 2012; Published September 5, 2012 Copyright: ß 2012 Arsenovic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research presented in this manuscript was funded by a grant from the Jeffress Memorial Trust (grant number J-925), and start-up funding from James Madison University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Tim A. Bloss is an author on patent# 20060199781 ‘‘Assays based on BTF3 activity’’. * E-mail: [email protected] Introduction chaperones prevent inappropriate associations between secondary structures and allow for the formation of functional domains as the To ensure survival, all organisms must tightly control the peptide is released into the cytosol. Translational chaperone synthesis and folding of proteins. To this end, specialized proteins interaction may also be important for protein localization, called chaperones regulate de novo protein-folding during and after specifically to organelles such as the ER and mitochondria. The translation, and recuperative protein-folding during denaturing translational chaperone system is best understood in Saccharomyces stress. Disruption of protein folding underlies the pathologies of cerevisiae, where the heat shock protein (HSP) 70/40 triad complex most, if not all, neurodegenerative disorders, including Alzhei- and the NAC associate with growing peptide chains at the mer’s, Huntington’s, and Parkinson’s diseases, as well as amyo- ribosome [6]. trophic lateral sclerosis (ALS) [1,2]. During disease development, The HSP 70/40 triad is a complex of three chaperones, two of the accumulation of improperly folded or aggregation-prone which form the ribosome-associated complex (RAC) that binds proteins leads to the formation of toxic oligomers that can trigger directly to the ribosomal complex, while the third, Ssb, binds both neuronal cell death [3]. To prevent protein aggregation, cells the ribosomal complex and the nascent peptide chain [7]. The initiate stress responses to forestall the accumulation of misfolded NAC consists of two subunits, a and b, both of which bind to the proteins. These responses decrease translation, increase turnover, polypeptide; bNAC also binds to the ribosomal complex [8,9]. and manage existing proteins that might otherwise aggregate [4]. Previous studies in yeast associate the NAC with promoting Misfolded protein management requires increased availability of protein localization away from the ER and to the mitochondria chaperones, which inhibit aggregation through direct interaction during translation, while more recent evidence suggests the NAC with their targets [5]. is also functionally connected to the HSP 70/40 chaperone Control of protein-folding begins at the ribosomal complex network and plays an important role in proper protein folding during translation. Highly conserved chaperones, known as the [10,11]. Deletion of NAC in Ssb-deficient yeast results in translational chaperone system, interact with both the ribosomal complex and the nascent polypeptide as it emerges. By doing so, PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44038 C. elegans NAC and the Unfolded Protein Response significant growth defects and accumulation of misfolded protein and theorized to be the result of autophagy associated with [12]. postmitotic cellular aging [23]. Although the NAC and the RAC are conserved throughout eukaryotic evolution, it is not clear that their chaperone functions Results in yeast are conserved in more complex organisms [12–14]. Our studies examine whether the NAC functions as a chaperone in C. Depletion of the NAC modulates stress response, elegans, which is genetically tractable much like yeast, but mortality and movement in the presence of heat substantially more complex with regards to stress response. For To characterize the putative chaperone role of the NAC, we example, eukaryotes respond to the accumulation of misfolded examined the ability of NAC-depleted animals to cope with protein in the ER with the unfolded protein response (UPR), a protein-denaturing heat stress. We reasoned that if the NAC is a mechanism that engages pathways involved in managing mis- component of a translational chaperone system, removal of either folded protein stress. In yeast, the UPR is mediated by a single NAC subunit (ICD-1 or -2) would exacerbate misfolded protein transmembrane ER protein, Ire-1. However, C. elegans contains accumulation during heat stress, and decrease worm survival. three transmembrane ER proteins, Ire-1, ATF6, and PERK/ Synchronized worms treated with icd-1 or icd-2(RNAi) were PEK-1, which independently or cooperatively initiate the UPR, exposed to chronic heat stress, and survival was measured by the and have conserved mammalian homologues [15]. Activation of presence of pharyngeal pumping. Unexpectedly, animals depleted these misfolded protein sensors can initiate a range of responses to of ICD-1 or -2 were not more sensitive to heat stress relative to maintain cell viability and functionality until acute stress is wild-type controls, as measured by mortality rates (Figure 1A). resolved and proteins are refolded properly. These responses ICD-2-depleted worms were instead more resistant to death than include the attenuation of protein translation, stimulation of either ICD-1-depleted or wild-type animals. To determine if the protein degradation, increase in ER and Golgi biogenesis, and up- loss of the NAC affected the vitality of worms under heat stress, the regulation of chaperone expression [16]. If misfolded protein stress movement of NAC(2) worms was measured relative to wild-type overwhelms the cell’s ability to manage it, the UPR engages in the presence of heat. Animals with decreased levels of ICD-1 or apoptotic pathways, which will kill the cell, or autophagic -2 were more mobile in these assays, with differences in robustness; pathways, which can also lead to cell death. The ability of the worms lacking ICD-2 showed the greatest movement, followed by UPR to kill chronically stressed cells links it to disease pathology, ICD-1-deficient and wild-type animals (Figure 1B). Movement was specifically proteopathic neurodegenerative diseases [17]. With strictly defined as an escape response from the touch of a metal pick, but in many cases, icd-1 and icd-2(RNAi)-treated worms were this in mind, the role of the NAC in stress response is particularly quite functional and often moving and feeding in the absence of interesting in C. elegans, a model organism with a well-defined and prodding, while their wild-type counterparts were immobile. precisely lineaged nervous system that allows for the study of NAC Taken together, these results indicate that depletion of the NAC chaperone function in neurons [18]. Induction